In this project we will use state-of-the-art technologies to address fundamental questions of cell lineage commitment, segregation and morphogenesis in mammals, using the mouse as a model. This proposal focuses on the gut endoderm, an embryonic tissue that gives rise to the major cell types of many internal organs, including the thyroid, thymus, lung, stomach, liver, pancreas, intestine and bladder. A rigorous understanding of normal gut endoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating gut endoderm and its derivative tissues, should underpin logical efforts to understand disease progression and design new therapeutic strategies for these vital organ systems. The prevailing view of germ layer formation in mammalian embryos is that the gut endoderm, along with the ectoderm and mesoderm, derives solely from the pluripotent epiblast during the process of gastrulation. Moreover, while extra-embryonic tissues interact with the epiblast to establish the body axes, they contribute solely to extra-embryonic structures, such as the yolk sac and placenta. Our studies challenge this view, and inform the hypotheses being tested in the Specific Aims of this project. The broad aim of this project is to use a combination of molecular, embryological and live imaging techniques to determine the fate of the visceral endoderm a presumed extra-embryonic tissue, and investigate its role(s) in the morphogenesis of the mammalian gut endoderm. In Specific Aim 1, we will determine if a lineage relationship exists between the visceral endoderm and the gut endoderm tissues of the fetus and adult mouse. In Specific Aim 2, we will elucidate the mechanisms that drive gut endoderm morphogenesis. Specific Aim 3, investigates the sequence of events leading to the organization of extra-embryonic (visceral) endoderm cells around midline signaling centers, and test the hypothesis that this arrangement is central to midline formation and/or function. Our findings are also relevant to stem cell differentiation for cell-based therapies and disease modeling because, current protocols for the in vitro differentiation of liver lung, pancreas and other endodermal cell types assume an epiblast-only origin and specifically select against the extra-embryonic endoderm that forms prior to gastrulation.